Various attempts have been made using microfluidics for the continuous separation of cells or microparticles. Some of the approaches combine microfluidics with an externally applied force field. For example, electrical, magnetic, optical, and acoustic-based forces have been attempted to separate particles. Still other approaches are based on the passive hydrodynamics created in microchannels. For example, various filters (e.g., weir-type, cross-flow type) and membranes have been proposed that operate based on size-exclusion principles. For example, Takagi et al. have developed a continuous particle separation technique that uses a microchannel having asymmetrically arranged multiple branch channels. See Takagi et al., Continuous particle separation in a microchannel having asymmetrically arranged multiple branches, Lab Chip, July; 5(7) 778-84 (2005). This method improves the separation scheme of pinched flow fractionation (PFF), which uses laminar flow within a microchannel.
Yamada et al. have proposed a microfluidic device for the continuous concentration and classification of particles using hydrodynamic filtration (HDF). This method uses various side channels to align particles along the wall of a microfluidic channel. Additional downstream selection channels are used to selectively extract different particles from the main channel. See Yamada et al., Hydrodynamic filtration for on-chip particle concentration and classification utilizing microfluidics, Lab Chip, November; 5(11): 1233-39 (2005). Choi et al. have developed a microfluidic separation and sizing technique for microparticles that uses hydrophoresis, the movement of suspended particles under the influence of a microstructure-induced pressure field. By exploiting slanted obstacles in a microchannel, one can generate a lateral pressure gradient so that microparticles can be deflected and arranged along the lateral flows induced by the gradient. See Choi et al., Continuous hydrophoretic separation and sizing of microparticles using slanted obstacles in a microchannel, Lab Chip, July; 7(7): 890-97 (2007).
Huang et al. have proposed a continuous particle separation method through deterministic lateral displacement (DLD). See Huang et al., Continuous Particle Separation Through Deterministic Lateral Displacement, Science, Vol. 304 No. 5673 pp. 987-990 (May 2004). This technique makes use of the asymmetric bifurcation of laminar flow around obstacles. A particle chooses its path deterministically on the basis of its size. Other methods are based on centrifugal separation. For instance, Ookawara et al. reported on the use of 200 μm×170 μm microchannels with semicircular radius of 2 mm for centrifugal separation where slurry particles are directed into one arm of a bifurcation channel. See Ookawara et al., K. Feasibility Study on Concentrator of Slurry and Classification of Contained Particles by Micro-Channel, Chem. Eng. J., v. 101, 171-178 (2004). More recently, Di Carlo et al. have developed an inertial focusing, ordering, and separation technique that orders particles in a controlled manner within a microfluidic channel. See Di Carlo et al., Continuous inertial focusing, ordering, and separation of particles in microchannels. PNAS, 104, 48, 18892-18897 (2007).
Shape, however, has rarely been considered in most of these integrated separation techniques, generally using the particle size, deformability, density, electric or magnetic characteristics or even its surface molecules to separate the particles while assuming cells and particles are spherical. Centrifugation, which is the macro-scale conventional technique for micro-particle separation, has been lately considered for shape-separation of spheres and rods. See Sharma et al., Shape separation of gold nanorods using centrifugation. PNAS, 106, 13, 4981-4985 (2009). Only recently, hydrodynamic filtration (HDF), deterministic lateral displacement (DLD) and dielectrophoresis (DEP) have begun considering shape as a criterion of separation in microsystems. Beech et al. first introduced the shape-based sorting with DLD technique, showing that non-spherical particles can be oriented in DLD devices via controlling device depth resulting in different effective dimensions to the pillars network. See Beech et al., Shape-based particle sorting—A new paradigm in microfluidics, Proc. Micro Total Analysis Systems, Jeju, Korea, 800-802 (2009). More recently, Sugaya et al. investigated the applicability of HDF for shape-based separation and demonstrated a difference in the separation behaviors of spherical and nonspherical particles at a branch point and used this technique for sorting budding/single cells from a yeast cell mixture. See Sugaya et al., Observation of nonspherical particle behaviors for continuous shape-based separation using hydrodynamic filtration, Biomicrofluidics, 5, 024103 (2011). Similarly, Valero et al. validated the shape-based sorting of yeast by balancing opposing DEP forces at multiple frequencies. See Valero et al., Tracking and synchronization of the yeast cell cycle using dielectrophoretic opacity, Lab Chip, 11, 1754-1760 (2011).
HDF and DLD are continuous and efficient techniques but both require low flow rates (2-3 μL/min and 60 mL/min, respectively) and high dilution factors, consequently offering a low throughput. These techniques also require accurately defined fabrication processes and complex designs, since the features that are necessary to guarantee the separation (pillar networks for DLD, highly-parallelized channels for HDF) have to be precisely designed (<1 μm-resolution). On the other hand, DEP requires the integration of active elements and a precise and reproducible control of the buffer conductivity between each experiment, which also complicates its integration in a whole-integrated microsystem. DEP based solutions require additional integration of active elements and a precise and reproducible control of the buffer conductivity between runs which makes DEP-based devices complicated and costly.